At the molecular level, multiple factors participate in the development of PMOP, interacting with each other and regulating bone metabolism in a complex regulatory network. Estrogen deficiency is the pivotal factor in the development of PMOP, which causes to the disorder of bone metabolism. Bone resorption is greater than bone formation, and eventually osteoporosis is caused [22]. Compared with hormone replacement therapy, traditional Chinese medicine, as a welcome alternative therapy, has greater advantages possessing many merits of simplicity and low side effect. Modern research have found that traditional Chinese herb contains various bioactive ingredients, which can act on multiple targets through multiple pathways, validating the holistic view of traditional Chinese medicine and the theory of treatment based on syndrome differentiation [23]. Compared with the “single component-single target-single pathway” research model of traditional pharmacology, network pharmacology combines the concepts of bioinformatics and multi-directional pharmacology to analyze the relationship between biological systems, medicines, and diseases from a network perspective. It has opened a new research model of traditional Chinese medicine from empirical medicine to evidence-based medicine [24].
Key active ingredient of ZYTLF for anti-PMOP
According to the network analysis results of the "herb-active ingredient-overlapping gene", the key active ingredients of ZYTLF are quercetin, kaempferol, luteolin, rhubarbin, formononetin and other flavonoid compounds. At the same time, they are also phytoestrogens, which has two-way regulating effects. It exerts the role of estrogen on target organ when estrogen deficiency occurs in body, and plays the role of anti-estrogen when the level of estrogen increases. It can alleviate a series of clinical symptoms caused by the postmenopausal estrogen deficiency and avoid the occurrence of tumors in the reproductive system caused by estrogen replacement therapy [25]. Quercetin with the highest degree value in the network is a typical flavonoid compound. It has multiple pharmacological effects, including scavenging free radical, anti-cancer, anti-infection, and protecting cardiovascular [26-29]. Quercetin has also been proven to be an effective component against osteoporosis, with the dual effects of inhibiting osteoclastogenesis and osteoblast differentiation. Zhixing Li found that quercetin can relieve osteoporosis symptoms in ovariectomized rats, possibly by up-regulating ALP gene expression and inhibiting JNK, ERK, and p38 MAPK signaling pathways [30]. Quercetin can also directly or indirectly down-regulate the expression of RANKL, inhibit osteoclast differentiation, reduce bone resorption, and stimulate osteoblast activity through estrogen signaling pathway and ERK signaling pathway [31]. Kaempferol has also been shown to have bone protective effects on ovariectomized rats [32], possibly through estrogen receptor, MAPK, NF-κB and other signaling pathways [33]. Luteolin can prevent bone loss after osteoporosis by inhibiting osteoclast differentiation.
Key Genes of ZYTLF for Preventing PMOP
Combined with the analysis of PPI network and key gene network, TNF, JUN, MAPK8, AKT1, IL-6, MMP9, PTGS2, MAPK1, CASP3 were of great importance and all related to the inflammatory response process. Actually, increasing studies have found that the reduction of estrogen levels in postmenopausal women can stimulate the immune system to produce a large number of osteoclastogenic factors, which in turn activates related signaling pathways, further aggravating bone loss [33]. Zha Li and other scholars have found that postmenopausal women with osteoporosis have significantly increased levels of TNF-α. In-vitro experiments shows that TNF-α and RANKL synergistically enhance bone resorption of osteoclasts through NF-κB and PI3K/Akt signaling pathways [35]. TNF-α and IL6 play an important role in the immune response and bone metabolism, mainly affecting the differentiation and proliferation of osteoclasts by regulating complex mechanisms, and they are important pathogenic factors for immune-mediated bone diseases [36]. Studies have shown that increased levels of inflammatory factor TNF-α in the serum of postmenopausal women may be one of the important causes of osteoporosis. TNF-α can activate the RANK/RANKL signaling pathway and induce the formation of osteoclasts [37]. We speculates that elevated TNF-α level in the serum of postmenopausal women with osteoporosis may be related to estrogen deficiency.
The signal pathways ZYTLF in preventing PMOP
The enrichment analysis of KEGG pathways for the key genes of ZYTLF in preventing PMOP showed that 15 signaling pathways were related to the development and progression of PMOP, including hormone pathways and inflammation-related pathways. Hormones in postmenopausal women, including estrogen [38], parathyroid hormone [39], and prolactin, if not normally secreted could affect bone metabolism [40]. Estrogen bound with the estrogen receptor in osteoblasts and osteoclasts to act on the OPG/RANK/RANKL signaling pathway, which further promoted OPG secretion, down-regulated the expression of RANKL, and inhibited the formation of osteoclasts [41]. After the combination of estrogen and estrogen receptor, it could also regulate the expression of various target genes through the estrogen signaling pathway, thereby activating downstream PI3K/Akt, MAPK, WNT and other signaling pathways to promote the differentiation and proliferation of osteoblasts [42-43]. The TNF signaling pathway had the largest number of enriched genes, which might be the key signaling pathway for ZYTLF to prevent PMOP. The TNF signaling pathway was mainly opened by TNF-α and interacted with multiple signaling pathways to synergistically inhibit osteoclast differentiation and bone resorption function [44].
Verification of molecular docking
In this study, network pharmacology was applied to elaborate on the potential anti-PMOP effect of ZYTLF and this effect was visualized by molecular docking for verification. In the result, ESR, TNF and other 21 proteins can combined with the top 10 active compounds, which have a significant role in regulation of the 23 proteins associated closely with PMOP. The result proved that ZYTLF could exert the treatment of PMOP by activating those receptors in the estrogen signaling pathway and TNF signaling pathway. It is consistent with the result of a vitro study showing that quercetin protected against TNF-α induced impairments in bone marrow mesenchymal stem cells[45]. In another study, quercetin can exert dual effect on estrogen, displaying anti-estrogenic effect at low dose (10mg/kg/day), and exaggerated estrogenic activities at high dose (100mg/kg/day)[46].
However, there are several limitations in this study. First, the compatibility and dosage of the formula are not taken into consideration. Second, the screened active components are different from the actual components in blood. Third, even if the results of network pharmacology and molecular docking simulation were combined, we still could not completely understand the accurate therapeutic mechanism of ZYTLF. Therefore, further experiments need to be conducted combining with Western Blot, Rt-PCR and multi-omics technology, etc. However, the network pharmacology methodology can save our time and energy by predicting the active ingredients, targets and signal pathways, and verifying it with molecular docking simulation.